FUNCTIONAL PROTEOMICS OF LEUKEMIC MARROW STROMAL CELLS

描述（由申请人提供）： 蛋白质组学，这需要全球基因表达分析，被认为是一个 无偏见的发现驱动（而不是假设驱动）的方法， 蛋白质表达分析。拟议的长期目标 研究的目的是确定人骨髓（BM）基质细胞在 正常和白血病造血。本提案旨在：（1）推进 先前的一项重要发现表明，人类骨髓基质细胞代表了一种 独特的多分化间充质祖细胞类型（MPC）， 共表达多种间充质谱系标志物。(2)最近申请 建立了从白血病BM基质细胞培养物中纯化MPC的方法， 不含巨噬细胞和造血细胞。(3)研究MPC细胞特异性 蛋白质表达谱以及这些谱在造血干细胞中的变化 恶性肿瘤。(4)建立的原则和实践证明 与正常人和白血病人骨髓相关的高分辨率蛋白质组学 基质细胞具体的实验设计包括以下性能： 以下. R21阶段：建立大幅面2-D凝胶电泳系统（2-D PAGE）用于MPC蛋白的可再现分离。制备2-D PAGE蛋白 正常BM来源的MPC的图谱（未处理的和用代表性药物处理的 细胞因子）和来源于代表性白血病患者的MPC 条件（AML，CML，MM）。使用质谱法（MALDI-MS和/或Nano ESI MS/MS），鉴定约200种差异表达的MPC蛋白（即，那些 与2-D PAGE蛋白质图谱相比， 正常的，未受刺激的MPCs）。人类骨髓MPC-2DPAGE数据库的构建 并在WORLD-2DPAGE下发布，并链接到当前现有的2-D PAGE数据库。在平行研究中，确定约200个差异 通过独立的方法表达MPC蛋白，同位素编码的亲和标签 基于理论pI和MW，构建用于整合的ICAT鉴定的MPC蛋白的“虚拟”2-D图谱 2-D PAGE数据库。R33阶段：为机器人添加高通量机器人 建立了R21期高分辨率双向电泳蛋白质组学。在A上识别 相对大规模（约1，800）的MPC蛋白， 在不同细胞因子刺激后和白血病中表达 (AML、CML、MM）。通过以下方式促进对发病机制的理解 鉴定可能参与细胞信号传导的磷蛋白 途径。在平行研究中，通过ICAT鉴定约1，800种MPC蛋白 法我们计划识别总共约2，000个功能相关的BM 基质细胞蛋白质。更新WWW数据库，包括 具有病理改变表达的蛋白质。数据库将是一个 研究基础生物学的研究人员的宝贵资源， 造血和白血病发生和临床血液学家/肿瘤学家 和病理学家一样

DESCRIPTION (provided by applicant): Proteomics, which entails global gene expression analysis, is considered a nonbiased discovery-driven (as opposed to hypothesis-driven) approach to the analysis of protein expression. The long-term objective of the proposed research is to determine the role of human bone marrow (BM) stromal cells in normal and leukemic hematopoiesis. This proposal aims to: (1) Carry forward a critical previous discovery that shows that human BM stromal cells represent a unique pluridifferentiated mesenchymal progenitor cell type (MPC), coexpressing multiple mesenchymal lineage markers. (2) Apply a recently established method for purifying MPCs from leukemic BM stromal cell cultures, free of macrophages and hematopoietic cells. (3) Investigate MPC cell-specific protein expression profiles and how these profiles change in hematopoietic malignancies. (4) Establish the proof of principle and practice of high-resolution proteomics with relevance to normal and leukemic human BM stromal cells. Specific experimental design includes performance of the following. R21 Phase: Set up large-format 2-D gel electrophoretic system (2-D PAGE) for reproducible separation of MPC proteins. Prepare 2-D PAGE protein maps for normal BM-derived MPCs (untreated and treated with representative cytokines) and for MPCs derived from patients with representative leukemic conditions (AML, CML, MM). Using mass spectrometry (MALDI-MS and/or Nano ESI MS/MS), identify about 200 differentially-expressed MPC proteins (i.e., those that increased or decreased in intensity as compared to 2-D PAGE protein maps of normal, unstimulated MPCs). Construct a human BM MPC-2DPAGE database on WWW and publish it under WORLD-2DPAGE with links to the currently existing 2-D PAGE databases. In parallel studies, identify about 200 differentially expressed MPC proteins by an independent method, isotope-coded affinity tag (ICAT) labeling in conjunction with LC/MS/MS. Based on theoretical pI and MW, construct a "virtual" 2-D map of ICAT-identified MPC proteins for integration into 2-D PAGE database. R33 Phase: Add high-throughput robotics to the high-resolution 2-D PAGE proteomics established under R21 phase. Identify on a relatively large-scale (about 1,800) the MPC proteins that are differentially expressed following stimulation with different cytokines and in leukemias (AML, CML, MM). Facilitate understanding of the pathogenetic mechanisms by identifying the phosphoproteins potentially involved in cell signaling pathways. In parallel studies, identity about 1,800 MPC proteins by ICAT method. We plan to identify a total of about 2,000 functionally relevant BM stromal cell proteins by each method. Update the WWW database by including the proteins with pathologically altered expression. The database will be a valuable resource for researchers investigating the basic biology of hematopoiesis and leukemogenesis and for clinical hematologists/oncologists and pathologists alike.